US6537800B1 - Apparatus for automatically measuring minute membrane potential - Google Patents

Apparatus for automatically measuring minute membrane potential Download PDF

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US6537800B1
US6537800B1 US09/623,969 US62396900A US6537800B1 US 6537800 B1 US6537800 B1 US 6537800B1 US 62396900 A US62396900 A US 62396900A US 6537800 B1 US6537800 B1 US 6537800B1
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membrane
electrode
cell
reaction
electrode according
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Isao Karube
Takashi Saitoh
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PUBLIC UNIVERSITY Corp AKITA PREFECTURAL UNIVERSITY
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/84—Manufacture, treatment, or detection of nanostructure
    • Y10S977/849—Manufacture, treatment, or detection of nanostructure with scanning probe
    • Y10S977/86—Scanning probe structure
    • Y10S977/873—Tip holder

Definitions

  • the present invention relates to an apparatus for measuring minute membrane potential, which measures a difference in potential between areas separated by a membrane or different positions on a membrane, such as an interior and exterior of a cell membrane, as well as an electrode constituting this apparatus.
  • the barrier-free technology initially concentrated on electronics, machinery, and construction but is being gradually expanded; some recent studies for the barrier-free technology are directly related to medicine. Typical examples include research on advanced medical equipment such as functional artificial hands or legs which supplement the functions of the bodies of handicapped people or the like. Such equipment is now controlled using methods such as electromyographic detection or detection of motions of the eyes or tongue, but information obtained by these input means is quantitatively significantly insufficient in terms of the amount of information required for motor and sensory functions of living bodies. If, for example, a person has lost his hand or leg due to a traffic accident, or a war wound, he must have an artificial hand or leg.
  • artificial inner ear One of the most successful examples in the field of direct exchange of information between nerves and electronic equipment is equipment called artificial inner ear [Rehabilitation Medicine, 31, 233-239 (1994)]. If an auditory defect occurs such that a portion between the tympanic membrane and the inner ear is prevented from functioning, the artificial inner ear executes the function of this portion by directly stimulating auditory nerves in the cochlear organ.
  • the artificial inner ear processes a sound input through a microphone depending on the auditory characteristic of this person to generate a digital signal in order to electrically stimulate terminals of the auditory nerves, that is, cochlear nerves.
  • Spherical electrodes are generally arranged at about 22 locations inside the cochlea, where about 30,000 auditory nerves are located, to input a sound signal to the nerves through the electrodes. For some time after the artificial inner ear has been implanted, most subjects feel that they hear very strange sounds, but once information processing in the brain has been adapted to the artificial inner ear, the subjects can distinguish sounds. Analysis and improvements of electrodes for the artificial inner ear is a large research area.
  • a substantial reduction in input auditory information from an inherent input through 30,000 nerves to an artificial input through about 22 electrodes means that auditory information received by the brain also decreases down to 0.1% or less.
  • regeneration of the sound listening ability depends on whether the brain can supplement and understand a substantially reduced amount of information.
  • Several months are required to optimally adapt the auditory processing function of the brain to the artificial inner ear.
  • the artificial inner ear suggests possibilities of nerve interfaces.
  • the electrodes are installed in the cochlear organ, where nerve terminals are exposed and lie over several centimeters.
  • the nerve stimulating electrodes may be installed at nerve terminals exposed to the interior of the organ, so that it is not physically difficult to arrange the electrodes.
  • the reasons why the artificial inner ear has successfully been put to practical use include many appropriate conditions for transmission of signals to the nerves through the electrodes. In other words, it is virtually impossible to directly apply the electrode technology for the artificial inner ear to nerves other than the cochlear nerves.
  • the other aspect is based on a long-term prospect for application of the information processing ability of nerve cells to computers [Bioelectrochemistry and Bioenergetics, 29, 193-204 (1992)] [Brain Research, 446, 189-194 (1988)].
  • This approach pursues the possibilities of what is called “biocomputers” that use living cells as operation elements.
  • Needle-shaped electrodes for measuring nerves have been used since the initial period of research on nerves, but the study of microneurography was the first to record the action potential of a single human peripheral nerve in situ [Clinical Electroencephalogram, 25, 493-500, 564-571, 629-638 (1983)].
  • the needle point holder-shaped electrode is one of the aggregate types in which this electrode is formed at a tip of a needle or on a side thereof and which is struck to a severed nerve bundle or a tissue in the brain [IEEE Trans. Biomed. Eng., 41, 1136-1146 (1994)]. That is, the needle point holder-shaped electrode is obtained by three-dimensionally expanding the needle-shaped electrode using a micromachine technology.
  • the needle-shaped electrode records a faint extracellular current from a nerve that is accidentally located close to the electrode section.
  • the needle-shaped electrode can be used to measure a single nerve cell, this method is evidently not accurate enough to simultaneously measure a large number of nerves even if the degree of integration of needle-shaped electrodes is increased to enhance spatial resolution, because the relative distance between each nerve and the electrode depends on accidents.
  • the axon regenerated matrix electrode in 2 is a field that has been expanded since 1992 when Stanford University conducted a relevant study, and many reports have recently been made on this electrode [IEEE Trans. Biomed. Eng. 39, 893-902 (1992)] [IEEE Trans. Biomed. Eng., 41, 567-577 (1994)] [IEEE Trans. Biomed. ENg., 41 305-313 (1994)].
  • This electrode is obtained by integrating electrodes into a 16 ⁇ 16 matrix or the like with a hole formed in each electrode section. The electrode is sandwiched between severed portions of a nerve bundle to record an extracellular current from each nerve axon regenerated through the corresponding hole in the electrode.
  • the axon regenerated electrode is advantageous in that the axon and the electrode are physically stably joined together and that a signal from the nerve axon passed through the hole in the electrode can be identified and detected.
  • severing the nerve bundle obviously adversely affects the nerves, and the nerve axons regenerated through the holes in the electrodes amounts to only several percents of the entire nerve bundle.
  • the axon regenerated electrode is expected to have its degree of integration increased so as to detect current in each nerve to electrically stimulate each nerve. If, however, attempts are made to increase the degree of integration, the area of the electrode section per nerve must be reduced while the area required for a wiring section of the electrode must be increased. Consequently, the rate of opening necessarily declines to significantly affect the spatial resolution.
  • the flat electrode method in 3 is often used for basic studies for biocomputers [Bioelectrochemistry and Bioenergetics, 29, 193-204 (1992)] [Brain Research, 446, 189-194 (1988)].
  • various useful information has been obtained on the shape and material of a substrate to which a cell or an axon adheres and on an applied voltage.
  • a problem of this method is a small contact area between the electrode and the cell.
  • a sufficient voltage and a sufficient contact area between the electrode and the cell are required to generate an action potential from the cell.
  • Application of a high voltage results in electrolysis of moisture, which is essential for a cell environment when the voltage exceeds about 1.3 V. Thus, the applicable voltage is limited.
  • a common disadvantage of these existing nerve interference is that they use the extracellular electrode to measure an electromotive force of about several ⁇ v induced by an action potential in the nerve based on a minor change in the concentration of ions outside the cell membrane.
  • the electrode picks up extracellular currents from cells in the neighborhood.
  • the electrode when the nerve is electrically stimulated to input a signal thereto, the electrode must provide a current much higher than an extracellular current from the nerve, thereby preventing measurements by other electrodes in the neighborhood.
  • the essence of nerve information is a membrane potential pulse from the nerve cell having a variation of about 100 mV. Accordingly, appropriate electrodes for use in exchanging information between the nerve and the equipment must be able to contact hard with the cell membrane or to be inserted through the cell membrane in order to measure variations in membrane potential.
  • the above-described interfaces using the extracellular electrodes can obtain a certain amount of information from the nerve and stimulate the nerve. None of these electrodes, however, has an enough spatial resolution to join to each nerve axon on a one-to-one correspondence, the nerve axon constituting a fiber for transmitting information. This is essentially disadvantageous in mutually separating nerve signals. Except for the central and terminal portions, the entire nerve axon has a mixture of inputs from a sensory organ to the center (afferent nerve fibers) and outputs from the center to a muscle or the like (efferent nerve fibers). Thus, when these interfaces have a low spatial resolution, input signals from the sensory organ and output signals to the muscle or the like are likely to be crossed. When nerve signals obtained are resolved and separated into signals for the individual nerves, a problem occurs even with the matrix electrode, which appears to have been most successful among the interfaces and which have been experimented in the situ systems.
  • the current electrodes that can input and output information to and from the individual nerve cell through the cell membrane include minute electrodes and patch lamp electrodes both used for electrophysicological experiments.
  • a tip portion of the electrode which contacts with the cell is thin and has a diameter between 200 nm and 2 ⁇ m, it is essential to integrate these electrodes together because their main body is composed of a glass tube of diameter several millimeters.
  • the electrode When the electrode is connected to the cell, it may destroy the cell membrane unless vibration of a measuring system is minimized.
  • a physical shearing force must be applied to the electrode to destroy the cell membrane. If the electrode uses as a support, a material of a high physical strength such as glass, the cell membrane can be destroyed by applying a physical shearing force to the electrode. This method, however, is inappropriate for minute metal electrodes in terms of structure and strength.
  • the existing cell membrane destroying method using only a physical shearing force is not the best method because the membrane is shaped for a high fluidity. That is, a sharp electrode alone cannot always penetrate the cell membrane. It is particularly difficult to insert the electrode into a cell in the order of several tens of micrometers simply by physically pressing the electrode into the cell.
  • a major reason why the patch electrode [Nature, 260, 799-802 (1976)][Pflugers Arch., 391, 85-100 (1981)] has been designed is that even the minute glass electrode, which is relatively strong, has the above problem; it is thus important to improve the electrode inserting method.
  • Activated oxygen such as singlet oxygen or superoxide radicals peroxidizes unsaturated phospholipid in the cell membrane through a chain reaction.
  • the cell has an oxidation defending mechanism such as ⁇ -tocopherol (vitamin E), which is an agent for capturing radicals in the membrane), or L-ascorbic acid (vitamin C) or superoxide dismutase (SOD), which is a water-soluble antioxidant, to resist oxidation.
  • vitamin E ⁇ -tocopherol
  • vitamin C L-ascorbic acid
  • SOD superoxide dismutase
  • Photosensitizers Molecules that generate activated oxygen to trigger this lipid chain peroxidation reaction when irradiated with light are called “photosensitizers (PS)”.
  • General photosensitizers include rose bengal and porphyrin.
  • the cain peroxidization reaction has only to be effected on a minimum area of a target cell surface for a short period of time. Moreover, if the membrane is damaged due to the peroxidation reaction during an electrode inserting operation, it is expected to be repaired by the above-described antioxidation system after the insertion.
  • the inventors have focused on a phospholipid radical chain peroxidization reaction as an example of destruction of the cell membrane using a method other than the physical shearing force.
  • Activated oxygen such as singlet oxygen or superoxide radicals peroxidizes unsaturated phospholipid in the cell membrane through a chain reaction.
  • the cell has an oxidation defending mechanism such as ⁇ -tocopherol (vitamin E), which is an agent for capturing radicals in the membrane), or L-ascorbic acid (vitamin C) or superoxide dismutase (SOD), which is a water-soluble antioxidant, to resist oxidation.
  • vitamin E ⁇ -tocopherol
  • vitamin C L-ascorbic acid
  • SOD superoxide dismutase
  • Photosensitizers Molecules that generate activated oxygen a trigger this lipid chain peroxidization reaction when irradiated with light are called “photosensitizers (PS)”.
  • General photosensitizers include rose bengal and porphyrin.
  • the chain peroxidization reaction has only to be effected partly on a target cell surface, that is, in a minimum area thereof for a short period of time, in order to denature the membrane. Moreover, if the membrane is damaged due to the peroxidization reaction during an electrode punching operation, it is expected to be repaired due to the fluidity of the membrane itself or by the above-described antioxidation system after the punching.
  • the inventors applied 5′5′′-bis(aminomethyl)-2,2,′:5′2′′-terthiophene dihydrochloride (BAT), one of the photosensitizers, to surface membranes of cultured cells PC12 from the nerve system.
  • the photosensitizer is a membrane denaturant that can be controlled by means of light irradiation.
  • Membrane resistance was measured to determine that the photosensitizer, as activated when the entire cell is irradiated with light, raises the membrane resistance, that is, ion permeability of the membrane.
  • the inventors have also clarified that the amounts of light and photosensitizer can be controlled to control a change in membrane resistance caused by light irradiation, to three levels.
  • the inventors have found a feature of this method that the ion permeability of the membrane recovers to its state prior to destruction in about 30 seconds under preferred conditions.
  • the inventors attempted to apply it to a microinjection process.
  • an injection liquid containing the water-soluble fluorescent dyeing reagent Lucifer Yellow CH (LY) was prepared and whether or not the LY could be injected into the PC12 cell was used as an index for determining whether or not the injection was successful.
  • an electric manipulator was used to automate the injection process to minimize the artificial effects on evaluation of the success rate.
  • the injection success rate was about 80%. In other control examples, the rate was about 0 to 10%. Therefore, membrane denaturing has been confirmed to significantly improve the injection success rate.
  • the cell survival rate was compared between the photosensitive process and the normal process.
  • Photosensitized cells exhibited a survival rate of about 90% for three to six days, which is significantly higher than that of normally processed cells, that is, about 10%.
  • the combination of the photosensitizer with light has been shown to serve to appropriately punch the membrane. That is, conditions for repairing the membrane without killing the cells can be easily determined depending on the level of membrane destruction.
  • a membrane destroying member with a membrane destroyer such as the photosensitizer applied to a support and then bring the membrane destroying member in contact with the membrane.
  • the inventors further produced a new member by providing a scanning probe of an atomic force microscope with an electrode and applying 5′5′′-bis(aminomethyl)-2,2′;5′2′′-terthiophene dihydrochloride to a probe section.
  • a scanning probe of an atomic force microscope with an electrode and applying 5′5′′-bis(aminomethyl)-2,2′;5′2′′-terthiophene dihydrochloride to a probe section.
  • the inventors attempted to achieved a spatial resolution of 20 ⁇ m using as an representative example of a nerve interface, one having such a specific target size of its own that it can be applied to mammalian peripheral nerves.
  • the value of 20 ⁇ m was set because peripheral myelinated nerves (A fibers) that carry out normal muscle control and sense transmission have a diameter between 1 and 22 ⁇ m.
  • the inventors further selected a rate phechromocyte PC12 cell as a nerve model.
  • the PC12 cell was established by Greene and Tischler in 1976 [Proc. Natl. Acad. Sci. USA 73, 2434-2428 (1976)]. This call is characterized to differentiate into a cell similar to a nerve when a nerve growth factor (NGF) is added thereto and is commonly used as a nerve cell model.
  • NGF nerve growth factor
  • An image of the cell can be picked up to specify an electrode inserting position.
  • the electrode can be manipulated at a spatial resolution in the order of nanometers.
  • a probe section of a commercially available ATM probe is shaped like a rectangular pyramid of base and height each 10 ⁇ m, so that the target spatial resolution of the 20 ⁇ m can be sufficient achieved even with modifications required to provide the probe with electrode.
  • Data obtained by the AFM probe electrode can be used to develop a nerve interface using a membrane-penetrating metal electrode.
  • the inventors have thus designed and developed a method for applying a cell membrane destroying technology using a method other than the physical shearing force, to insertion of the electrodes.
  • the inventors have succeeded in allowing the electrode to easily penetrate the membrane by coupling an electrode inserting operation to a cell membrane destroying operation using a compound that generates an activated oxygen species.
  • the inventors succeeded in producing an atomic force microscope actually equipped with a cell membrane destroying probe electrode, selecting an electrode installing portion while measuring the shape of a surface of the cell, and then inserting the electrode into the cell membrane.
  • Those skilled in the art can integrate a number of electrodes together as appropriate using the above single electrode as a prototype.
  • the present invention includes:
  • an electrode comprising an insulated support, a conductive pattern formed on a surface of the insulated support, an insulator formed on the conductive pattern in such a manner that a portion on the conductive pattern which comes in contact with at least a membrane after the membrane has been penetrated can be insulated, and a membrane denaturation reaction prompting portion formed in the portion coming in contact with the membrane or in a neighborhood thereof and having a membrane denaturing force other than a physical shearing force,
  • the electrode according to (1) wherein the insulated support comprises a particular support and an insulating layer covering a surface of the support,
  • an electrode including a support comprising silicon processed by means of etching, wherein gold (Au) with a thickness of 220 nm is plated on a bottom surface (a measuring surface) of the support, areas of the electrode other than a measuring metal terminal and an equipment-connected metal terminal are insulated and covered by silicon dioxide with a thickness of 100 nm, and 5′5′′-bis(aminomethyl)-2,2′:5′2′′-terthiophene is applied to an electrode section,
  • an interface type minute membrane potential measuring apparatus comprising the electrode and the potential measuring device according to one or more of (1) to (12).
  • the electrode provided by the present invention comprises the membrane denaturation reaction promoting portion having a membrane denaturing force other than the physical shearing force.
  • the electrode is connected to the position controlling device and, more preferably, it contacts therewith at such an arbitrary pressure that a surface of the membrane is not destroyed.
  • the electrode When the electrode is based on a common one including a support, a measuring metal terminal, an insulating portion and can retain electrode functions, it can be composed of common materials.
  • the support may preferably composed of silicon, glass, or the like.
  • the material of the conductive pattern may be gold (Au), platinum (Pt), or other metal generally used for patterning in the field of electronics.
  • An example of the insulator used in the insulating portion includes silicon dioxide or silicon nitride.
  • the electrode may have additional functional, for example, an electrode position indicating function based on a fluorescent coating or an extended lifetime resulting from coating with an antiseptic unless the electrode functions are degraded.
  • the membrane denaturing portion of the electrode having the membrane denaturation reaction promoting portion refers to a portion that comes in contact with a particular portion of the membrane which is denatured or destroyed.
  • the membrane denaturation reaction may be a chemical reaction including a reaction using radiation, laser beams, or the like, an enzymatic reaction using lipase or protease, or a chained peroxidization reaction of membrane components started by a direct or indirect generation reaction of an activated oxygen species.
  • An example of the chained peroxidization reaction includes a lipid peroxidization reaction of a living membrane, and, more specifically, includes generation of a radical, singlet oxygen, superoxide, or generation of hydrogen peroxide.
  • the electrode does not necessarily require a substance causing the membrane denaturation reaction to be applied or fixed to the membrane denaturation reaction prompting portion if radiation or the like is used, it may have a compound applied or fixed to a portion thereof, the compound including an enzyme or a compound that involves generation of activated oxygen and causing the membrane denaturation reaction.
  • the applied or fixed compound is a reaction precursor that causes the membrane denaturation reaction when subjected to a particular stimulus.
  • An example of the combination of the “particular stimulus” and the “reaction precursor” includes “light” and a “photosensitizer”for a light-induced electron transfer reaction, “radiation” and a “cell membrane and moisture molecules” for a radiative chemical reaction”, or a “change in electrode potential” and a “conductive polymer” for an electrode reaction.
  • the photosensitizer that promotes generation of singlet oxygen when irradiated with light includes methylene blue, rose bengal, chlorophyll (a common name), hematoporphyrin, posoralen, bilirubin, riboflavin, chlorophyll, or retinal, and the compound that generates activated oxygen on the electrode upon application of a potential includes methylviologen.
  • An example of the conductive polymer that is radicalized on the electrode when provided with a potential includes polyacetylene or polythiophen.
  • a thiophen trimer (terthienyl) is a natural photosensitizer obtained from a plant and can be used as a compound that can induce membrane destruction upon application of light or a potential.
  • the compound that catalyzes radicalization when irradiated with light may be semiconductor particles such as a ruhtenium (II) trisbipyridine complex or titanium oxide, or a starting agent that starts radical polymerization when irradiated with light may be used as a photosensitizer.
  • the photopolymerization sensitizer based on irradiation with ultraviolet rays may be a peroxide such as a benzoyl peroxide, an azo compound such as azobisisobutyronitryl, a carbonyl sulfur compound such as diacetyl, dibenzyl, etc.
  • diphenyl monosulfide diphenyl disulfide, dibenzoyl monosulfide, or dibenzoyl disulfide
  • a halogen compound such as CCl 4
  • a metallic salt such as FeCl 3
  • the level of membrane destruction based on lipid peroxidizing cannot be easily controlled based only on the quantitative ratio of the “particular stimulus” and the “compound activated by a stimulus” or the like, the level of destruction may be controlled by adding a substance such as lipid or an oxidation inhibiting substance that weakens the lipid peroxidization reaction or assists membrane repairs.
  • the electrode having the membrane denaturation reaction promoting portion as described above may contact with the membrane so as not to completely destroy it or may be used in such a manner that after the membrane has been denatured or destroyed, the electrode penetrates the membrane, which comes in close contact only with part of the insulating portion.
  • a specific example is an electrode including a support comprising silicon processed by means of etching, wherein gold (Au) with a thickness of 220 nm is plated on a bottom surface (a measuring surface) of the support, areas of the electrode other than a measuring metal terminal and an equipment-connected metal terminal are insulated and covered by silicon dioxide with a thickness of 100 nm, and 5′5′′-bis(aminomethyl)-2,2′:5′2′′-terthiophene is applied to an electrode section.
  • both types of electrodes may be connected to the position controlling device to control the position where the electrode is inserted into or contacted with the membrane or may have a function for measuring the shape of the surface of the membrane or a solid, or may be a scanning probe of the scanning probe microscope.
  • both electrode can preferably be used even if at least part of the measuring metal terminal is covered with an insulated or genetic film, unless the electrode functions are lost.
  • an interface type minute membrane potential measuring apparatus can be provided which includes a combination of a plurality of the above-described electrodes and a potential measuring device. This interface type minute membrane potential measuring apparatus can also be provided as a composite interface type minute membrane potential measuring apparatus including an additional normal electrode or normal metal terminal, or a combination with another potential measuring terminal.
  • all the above-described electrodes or potential measuring devices can be used as nerve interfaces having the electrode connected to the individual nerve to transmit information between electric information equipment and the nerve, so that they can each be provided as part of precision equipment for use in research such as basic analysis and research on the brain, clarification of living cell mechanisms, or analysis of functional electric stimuli.
  • Each of them can also be used as part of a highly integrated and very accurate invasive measuring type medical electrode or part of a connection and control device for living function substituting and supporting equipment such as an artificial organ.
  • any of the above-described electrodes or potential measuring devices can alternatively be used for part of a connection and control device for an artificial hand or leg comprising a joint or a sensory organ that can be controlled as in human bodies, or as part of a connection and control device between an artificial sensory organ (a visual or auditory sense) and a living body.
  • Any of the above-described electrodes or potential measuring devices can alternatively be used as a part of a brain function expanding apparatus for patients who have their brains damaged due to the Alzheimer's disease or the Parkinson's disease or as a part of a living function expanding apparatus for recovering a living function lost due to a congenital disease. Communication among living things may be extended by connecting the above-described interface type minute membrane potential measuring apparatus using the electrode not only to human beings but also to general animals or even plants to clarify living information processing mechanisms.
  • FIG. 1 shows a structural formula of a 5′5′′-bis(aminomethyl)-2,2′:5,′2′′-terthiophene dihydrochloride (BAT) dichloride salt.
  • FIG. 2 shows a membrane punching technique flow chart.
  • FIGS. 3A-3D show the relationship between a membrane destroying member having a cylindrical support and a membrane structure that is to be processed by the membrane destroying member.
  • FIGS. 4A-4D show an example of the relationship between a membrane destroying member having a sphere- or bead-shaped support and a membrane structure that is to be processed by the membrane destroying member.
  • FIGS. 5A-5D show an example of the relationship between a membrane destroying member having a rod-shaped support and a membrane structure that is to be processed by the membrane destroying member.
  • FIGS. 6A-6D show an example of relationship between a cylindrical membrane destroying member having a support for holding a liquid containing a membrane denaturation reaction promoter and a membrane structure that is to be processed by the membrane destroying member.
  • FIGS. 7A-7D show an example of relationship between a member destroying member having a support shaped like an atomic force microscope probe and a membrane structure that is to be processed by the membrane destroying member.
  • FIG. 8 schematically shows an apparatus for measuring intracellular potential and membrane resistance using a patch electrode.
  • FIGS. 9A-9C are graphs representing the relationship between the intensity of a light stimulus and variations in cell membrane potential or membrane resistance.
  • FIG. 10 is a flow chart of an example of photosensitized injection.
  • FIGS. 11A and 11B schematically show the example of photosensitized injection.
  • FIG. 12 shows a comparison of an injection success rate between a normal physical injection process and a new photosensitized injection process.
  • FIG. 13 shows a comparison of variations in cell survival rate after a cell injection process between the normal physical injection process and the new photosensitized injection process.
  • FIGS. 14A and 14B schematically show a minute electrode produced using a scanning probe of an atomic force microscope as a support.
  • An established nerve system cell line PC 12 as ganglion-like cells obtained from the adrenal medulla of a rat and which are used as a model of the central nerve.
  • a neurobasal medium (manufactured by GIBRO BRL) (pH 7:3) containing 10% of thermally inactivated equine serum, 5% of bovine fetus serum, 7.35 mg/l of L-glutamic acid, and 2 mM of L-glutamine was used to culture PC 12 cells in an atmosphere containing 95% of CO 2 .
  • the cells were subcultured by spraying a culture medium on the cells, peeling off the cells from a wall surface of a culture flask, centrifuging this 380-g flask for 5 minutes to bias the cells, scattering the cells into a culture flask of base area 25 cm 2 (manufactured by IWAKI GLASS Co., Ltd.) for 1 to 3 ⁇ 10 4 cells/cm 2 per ml, and changing the culture medium at the intervals of two or three days.
  • NGF mouse nerve growth factor
  • phosphate buffered saline PBS it was composed of 2.10 g/l of KN 2 PO 4 , 90.00 g/l of NaCI, 7.26 g/l of NaHPO 4 ,7H 2 , and a 1-N NaOH solution, with a pH adjusted to 7.4) was provided.
  • the NGF solution dispensed in this manner was added to a culture medium so as to reduce the concentration to 1/1,000, thereby differentiating the PC 12 cells.
  • the PC 12 cells were allowed to adhere lightly to a wall surface of a plastic bottle, where they were grown while forming a small cluster.
  • collagen coat dishes IWAKI Glass Co., Ltd.
  • the pnotosensitizer was composed of 5′5′′-bis(aminomethyl)-2,2′:5′2′′-terthiophene dihydrochloride (BAT), which is an ⁇ terthienyl derivative.
  • BAT 5′5′′-bis(aminomethyl)-2,2′:5′2′′-terthiophene dihydrochloride
  • This compound was synthesized in accordance with the article of Muguruma [J. Hetercyclic Chem., 33, 1-6 (1996)]and provided in a state of BAT dichloride salt.
  • FIG. 1 shows a structural formula for a BAT dichloride salt.
  • a thiophen oligomer having an aminomethyl group at a terminal thereof has a higher water solubility than any other derivative of the same kind due to this terminal aminomethyl group.
  • the water solubility varies depending on how the aminomethyl group is released.
  • the present BAT is characterized by having two types: one of them has a bivalent positive charge in an acid water solution and is easily soluble therein, while it has a univalent positive charge in a water solution with a biologically suitable pH range (near 7.4) and maintains a high water solubility; the other exhibits the same characteristic in the former solution but in the latter solution, has no charge and is likely to cohere into a colloid.
  • this molecule By irrigating the cell with the BAT-dispersed solution under the above pH condition, this molecule can be easily added to a surface of the cell.
  • the hydrophilic property of the BAT molecule is new and is absent from the other modified thiophen oligomers including an a terthienyl derivative as well as other molecules designed as conductive high-molecular monomers.
  • the patch clamp method which is used for electrophysiological experiments, was used to measure the potential between the cell membranes or ion current transmitted through the cell membrane.
  • the photosensitizer BAT was dispersed in a HEPES (25 mM, pH 7.4) buffer.
  • a dispersed solution to be locally added to a neighborhood of the cell using a micropipette had a BAT concentration adjusted to 2 mM, and a dispersed solution to be added to the entire irrigation liquid had a BAT concentration adjusted to 0.2 mM.
  • the cells were incubated in a culture medium for electrophysiological experiments at roam temperature.
  • the culture medium used for this experiment was composed of 124 mM of NaCl, 5 mM of KCl, 2.4 mM of CaCl 2 .2H 2 O, 1.3 mM of MgSO 4 .7H 2 O, and 10 mM of glucose, with a final pH adjusted to 7.4 by addition of NaOH.
  • the culture medium for electrophysiological experiments was replaced with a new at the intervals of 40 minutes at maximum.
  • the BAT was added so as to have a final Concentration of 49 ⁇ M.
  • the quantity of light applied was set at three levels: 0.47 J/cm 2 , 0.94 J/cm 2 , and 1.57 J/cm 2 .
  • a liquid in the patch electrode was composed of 132 mM of KCl, 8 mM of NaCl, 2 mM of MgCl 2 , 30 mM of HEPES, 4 mM of Na 2 ATP, 0.3 mM of GTP, and 0.5 mM of EGTA, with a final pH adjusted to 7.3 by addition of NaOH.
  • An excitation light source was a 50-mW and 363-nm argon ion laser including a confocal laser scanning microscope (CLSM) MRC-1000 UV (BIO-RAD laboratories) as standard equipment.
  • CLSM confocal laser scanning microscope
  • one-sixteenth (1 ⁇ 4 along each of the ⁇ and Y axes; 117 ⁇ 170 ⁇ m) of the full screen (about 470 ⁇ 680 ⁇ m of the microscope was scanned by the laser beams. This area was set to cover the entire patched target cell.
  • the laser beams had a 100% output of 50 mW.
  • the amount of irradiation time was selected from ⁇ fraction (1/16) ⁇ , 1 ⁇ 4, and ⁇ fraction (1/32) ⁇ seconds depending on a scan speed.
  • a filter was also used to reduce the amount of light.
  • a TTL signal operating in connection with an electrophysiological record is transmitted to the light source as required to synchronize the light irradiation with electric measurements.
  • the cell membrane potential had been maintained between ⁇ 80 and ⁇ 60 mV.
  • the patch electrode had a resistance between 3 and 4 ⁇ and was filled with the above-described intraelectrode liquid.
  • the cell membrane potential measuring amplifier was Axpopatch 1-D (Axon Instruments).
  • the membrane resistance was calculated from a change in membrane potential occurring when a 1-Hz rectangular-wave overpolarization current flowed through the membrane for 350 milliseconds.
  • the amount of, current supplied (0.1 or 0.15 nA) was selected so that the change in membrane potential would not exceed 30 mV when power was supplied. Under these experimental conditions, the PC 12 cell generated no action potential.
  • Measured potential and current values were analyzed using Axoscope ver. 1.1 (manufactured by Axon Instruments). Results are shown in FIG. 9 .
  • the axis of abscissa indicates an elapsed time (unit: second).
  • the axis of ordinate indicates the resistance of the cell membrane (unit: %) in the upper part and the potential thereof in the lower part, both values being normalized by setting corresponding values prior to irradiation equal to 140.
  • the membrane resistance reflects the ion transmission inhibiting capability
  • the membrane potential reflects the active ion transporting capability of the membrane effected by activities of various intermembrane ion transporting systems as well as the ion transmission inhibiting capability.
  • the amount of light applied was smallest for A (0.47 J/cm 3 ), the second smallest for B (4.94 J/cm 2 ), and the largest for C (1.57 J/cm 2 ).
  • A both cell membrane resistance and membrane potential varied slightly but not significantly after the light irradiation.
  • B the cell membrane resistance decreased and the membrane potential was depolarized several seconds after the light irradiation; this amount of time corresponds to an induction period. Under these conditions, the resistance and potential were also observed to recover to the corresponding values prior to the light irradiation 30 minutes later. This is because, after destruction, the membrane was repaired due to a biological reaction.
  • the cell membrane resistance and membrane potential were lost several seconds (also corresponding to an induction period), that is, about 8 seconds after the light irradiation and then remained at fixed values. This is because no repair reaction occurred after the membrane was destroyed, that is, irreversible membrane destruction occurred.
  • the water-soluble fluorescent pigment Lucifer Yellow CH (LY) was added to an injection liquid. After the injection process, this process was determined to be successful when a LA—induced yellow fluorescence was observed in the cell through a fluorescence microscope. Evaluation was made to determine how the success rate of injection of the LY into the cell varied depending on the presence of the photosesirizer BAT in the injection liquid or the presence of application of the BAT exciting light.
  • the LY is a less toxic fluorescent pigment used for microinjection and is characterized by shifting to a daughter cell upon cell division [Cell & Tissue Res., 234, 309-318 (1983)].
  • the LY is so water soluble and dispersible that it is also used as cell fluorescent label agent for the nerve system [Cell & Tissue Res., 254, 561-571 (1988)].
  • the LY is also characterized by shifting fast in several minutes between cells coupled together by means of a gap junction [“New Physiological System 7, Physiology for Generation and Differentiation, Chapter 4, Generation of Intercell Coupling, I. Electric Coupling”, Medical Publishing Company (1991)], which is a liquid-liquid junction the between cells.
  • the micromanipulator served to automate the injection operation to achieve a high reproducibility and the commercially available capillary was used, which is more uniform in shape than those made by the inventors, a statistical process could be carried out for the injection success rate.
  • the micromanipulator was installed in the fluorescent microscope (manufactured by Olympus Optical industry Co., Ltd. according to a specification for an IX70 fluorescent microscope).
  • the light source for exciting the photosensitizer was an ultraviolet light obtained by transmitting light through an ultraviolet exciting filter set (manufactured by Olympus Optical Industry Co., Ltd.; a U-MWU mirror unit), the light being emitted from a 100-W mercury lamp that was an epifluorescent light source built into the microscope.
  • the photosensitizer BAT was concentrated in the area of diameter 0.5 ⁇ m, while it was promptly diluted due to dispersion in areas other than the one contacted with the capillary.
  • the photosensitizing effect of the BAT is equal to that of 100
  • the photosensitizes BAT was synthesized and provided by Muguruma et al.
  • the reagents listed below were used far the experiments and were commercially available: sodium chloride (NaCl; manufactured by Rishida Chemistry Co., Ltd.), potassium chloride KCl, manufactured by Rishida Chemistry Co., Ltd.), disodium hydrogenphosphate (Na 2 HPO 4 ; manufactured by Wako Pure Chemical Industires Co., Ltd.), dipotassium hydrogenphosphate (KH 2 PO 4 ; manufactured by Wako Pure chemical Industires Co., Ltd.), and fluorescent marker Lucifer Yellow CH, Lithium salt (LY, ex. 482 nm, em. 536 nm; manufactured, by Molecular Probes).
  • Each component of the injection liquid was dissolved into pure water so as to prepare a composition having the following final concentrations: 100 ⁇ m of photosensitizes BAT, 40 ⁇ m of HCl, 2 mM of fluorescent marker LY, 8 g/l of NaCl, 0.2 g/l of KCl, 1.15 g/l of Na 2 HPO 4 , and 0.2 g/l of KH 2 PO 4 .
  • the injection target cells were the established nerve system cell line P 12 . These cells were obtained from the Cell Development Hank of the Physical and Chemical Research Institute and cultured in accordance with Example 2. The cells subjected to the injection process were subcultured in 35-mm collagen coated dishes (Iwaki Glass Co., Ltd.) at a cell density of 9,000 cells per dish. The cells were cultured by a carbonate dioxide gas incubator (manufactured by Forma Scientific) in an environment at a temperature of 37° C. and a humidity of 100% and containing 5% of carbon dioxide and 95% of oxygen.
  • a carbonate dioxide gas incubator manufactured by Forma Scientific
  • Hibernat A Media Hib-A; manufactured by GIBCO BRL [NeuroReport, 7, 1509-1512 (1996)]that are Hib-A culture media for the injection process and to which were added 10% of equine serum (manufactured by GIBCO BRL), 5% of bovine fetus serum (manufactured by Mitsubishi Chemistry Co., Ltd.; a serum obtained from a quasi-fetus of a Nakashibetsu cow), 7.35 mg/l of L-glutamic acid (manufactured by GIBCO BRL), and 2 mM of L-glutamine (manufactured by GIBCO BRL).
  • the equine serum was thermally inactivated by heating it at 56° C. for 30 minutes.
  • water was used which was purified by a pure-water-manufacturing-apparatus Biocel A10/Elix 10 (manufactured by MILLIPORE).
  • FIG. 10 An outline of the injection process procedure is shown in FIG. 10 . The following supplement this figure.
  • a limit position (Z limit) at which the tip of the capillary came closest to a surface of the dish had to be set so that the tip could be stuck into the cell during the injection.
  • the manipulator was set so that the Z limit position aligned with the position of a cell nucleus on the dish.
  • the capillary position was changed to one 30 ⁇ m above the Z limit.
  • the other conditions that is, an approach speed input value and an injection time were also changed to 700 ⁇ ms ⁇ 1 and 1.1 s, respectively (effective values were 1,000 ⁇ ms ⁇ 1 and 1.0 s, respectively).
  • the z limit position was adjusted so that under these conditions, the normal physical microinjection process could be achieved at a success rate of 80% or more for 10 or more cells. When the success rate was lower, the Z limit position was reset.
  • the capillary position was changed to 10 ⁇ m above the z limit.
  • the approach speed input value and the injection time were changed to 5 ⁇ ms 31 1 and 124 seconds, respectively (the effective value of the injection time was 120 seconds).
  • the positions of the cell and the capillary were adjusted so that the capillary tip was located at the center of the cell.
  • the epifluorescent light source was switched to the ultraviolet exciting (U-MWU mirror unit) filter set to apply ultraviolet rays.
  • the transmissive light source for observing the cells was interrupted.
  • the filter of the epifluorescent light source was switched from the one for ultraviolet light to the one for violet light (U-MWBV mirror unit) to apply the Lx exciting light in order to check whether the cell had been dyed with the LY. If the liquid is injected into a dead cell, the LY leaks quickly from the cell membrane and the fluorescence disappears. Thus, such cells were removed from the data.
  • the injection process was repeated.
  • the Hib-A culture medium for the injection process was removed from the dish, and the dish was washed twice using 1 ml of PBS and then returned to the 3 ml of NeuroBasal culture medium. Subsequently, the culture medium was changed in accordance with a normal procedure.
  • the injection had to be executed under the condition that the physical shearing force of the pipette tip did not contribute to the injection.
  • the adjustment of the arrival limit position (Z limit) of the capillary is important.
  • the approach speed was lowered to 7 ⁇ ms ⁇ 1 .
  • the capillary tip can reach the same position as in the successful injection, but it hardly penetrates the cell membrane. Under such an approach condition that the membrane could not be punched easily with the physical shearing force, injection success rates obtained using the photosensitizing mechanism were compared together. Results are shown in FIG. 12 .
  • the axis of abscissa indicates injection process conditions, while the axis of ordinate indicates the injection success rate (unit: %).
  • the injection success rate was between 0 and 10% at a capillary approach speed of 7 ⁇ ms ⁇ 1 , whether or not UV light was applied (when UV light was applied, the injection was successful with none of the 30 cells; 0%) (when no UV light was applied, the injection was successful with 3 of the 30 cells; 10%).
  • a cell survival rate after the injection process was compared between cells subjected to the normal microinjection process using the physical shearing force and cells subjected to the photosensitizing microinjection.
  • the LY was injected into the cell by means of microinjection. In a dead cell with its cell membrane collapsed, the LY disperses quickly to lose its color [Cell, 14, 741-759 (1987)]. Thus, using a rate at which the cell subjected to the injection process holds the LY, as an index indicating the cell survival rate, the survival rates 3 and 6 days after the injection process were compared to each other. Results are shown in FIG. 13 .
  • the axis of abscissa indicates the number of days passing after the injection process (unit: day), while the axis of ordinate indicates the survival rate (units %).
  • the survival rate was observed to be 30% or lower 3 days after the injection (LY: 17%; BAT+LY: 30%). No change was observed between the LY retaining rate at day 3 and day 6 for all of the three injection Conditions.
  • the cell survival rate was between 20 and 30% A cell survival rate of about 90% for the cells subjected to the photosensitizing injection indicates that this injection causes little damage to the cells.
  • the LY injected into the cell disperses quickly through the cytoplasm to dye the cells.
  • the LY disperses to the exterior of the cell 1 or 2 seconds after the injection.
  • This quick dispersion of the LY has been reported to also occur through a gap junction, which is a liquid-liquid connecting path [Cell & Tissue Res., 234, 309-318 (1983)] [d. Neurosci., 14, 3945-3957 (1994)].
  • the punched cell membrane is assumed to be re-blocked in the cell holding the LY after the injection process. This prompt repair of the membrane is also supported by the fact that the membrane potential and resistance recover within several minutes after light irradiation in Example 3.
  • the LY was injected into the cell by means of the photosensitizing injection.
  • the Lx can permeate not only into a cell into which it has been injected but also into adjacent cells coupled thereto by means of the gap junction, so that all these cells are dyed [Cell & Tissue Res., 234, 309-318 (1983)][J. Neurosci., 14, 3945-3957 (1994)].
  • One of the functions of the gap junction is an electric connection between the cells [New Physiological System 7, Physiology for Generation and Differentiation, Chapter 4, Generation of Intercell Coupling, I. Electric Connection”, Medical Publishing Company (1991)].
  • myocardial cells or the like contract synchronously all over the muscle tissue because a large number of muscle cells are electrically connected together via the gap junction.
  • the present photosensitizing injection is technically more advantageous than the normal microinjection based on the physical shearing force, in terms of damage to the cell caused by the injection.
  • a probe side (a measuring surface) of a commercially available monocrystal scanning probe processed by means of etching was plated with gold (Au) with a thickness of 220 nm by means of sputtering (Shibaura Production Company; sputter operation pressures 0.3 Pa; output: 100 W). Areas of the probe other than a measuring metal terminal and an equipment-connected metal terminal were insulated and covered by silicon dioxide of thickness 100 nm.
  • This scanning probe was installed in an atomic force microscope (Nanoscope III; manufactured by Digital Instruments), and the equipment-side metal terminal was connected to a negative electrode of an electric punching apparatus (Gene Pulser; manufactured by BIO-RAD laboratories).
  • a positive electrode of the electric punching apparatus was connected to a metal substrate (copper, platinum, or the like) on an AFM sample plate, and the probe, which had not had its insulation destroyed, that is, which was still insulated and coated by silicon dioxide, was brought in contact with the substrate.
  • a 3- ⁇ resistor was connected in series between the scanning probe in contact with the substrate and the electric punching apparatus to prevent the probe from being destroyed by an excessive current after the insulation had been destroyed.
  • a battery rating and voltage for the electric punching apparatus were set at 0.25 ⁇ F and 50 V, respectively, and electricity was instantaneously conducted between the substrate and the scanning probe section. This conduction caused the insulation at the tip portion of the probe to be destroyed to expose a metal layer from the tip portion. Thus, a minute Metal electrode was completed which had only the tip portion of the probe exposed as an electrode.
  • the Lip portion of the probe of the minute metal electrode was immersed in an acid cater solution containing 2 mM of BAT (pH 3.0). This operation caused the BAT to be adsorbed by and fixed to the electrode. Finally, the electrode was washed off in distilled water to remove an excess portion of the BAT.
  • the technique for controlling the membrane denaturation reaction without using the physical shearing force has been developed to enable the membrane to be denatured or punched more easily than before.
  • the use of the minute metal electrode facilitates the insertion of the electrode into the cell using the minute metal electrode, as is difficult in the prior art, thereby enabling the membrane potential in the cell to be easily measured.
  • the minute metal electrodes can be integrated together, so that a nerve interface far the barrier free technology can be developed.

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004053596A1 (de) * 2004-11-05 2006-05-11 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtungen zur Bearbeitung einzelner biologischer Zellen
US20070005117A1 (en) * 2005-06-30 2007-01-04 Fritsch Michael H Extra-cochlear implanted hearing aid device
US20070172600A1 (en) * 2003-04-25 2007-07-26 National Institute Of Information And Communication Technology Incorporated Molecular bonding method and molecular bonding device
US20070225776A1 (en) * 2006-03-22 2007-09-27 Fritsch Michael H Intracochlear Nanotechnology and Perfusion Hearing Aid Device
US20080014573A1 (en) * 2004-05-26 2008-01-17 Yunisoku Corporation Biosample Manipulation Apparatus
US20080220517A1 (en) * 1998-03-12 2008-09-11 Toudai Tlo, Ltd. Site-specific cell perforation technique
US20080302674A1 (en) * 2004-12-21 2008-12-11 Kyoto University Probe Unit, Apparatus for Identifying Nucleotide Region and Method of Identifying Nucleotide Region
US20140199765A1 (en) * 2011-05-24 2014-07-17 Brigham Young University Lance device and associated methods for delivering a biological material into a cell
US8909348B2 (en) 2010-03-30 2014-12-09 Domestic Legacy Limited Partnership Cochlear implant stabilization and positioning device
US10119151B2 (en) 2007-07-09 2018-11-06 Brigham Young University Methods and devices for charged molecule manipulation

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6686193B2 (en) 2000-07-10 2004-02-03 Vertex Pharmaceuticals, Inc. High throughput method and system for screening candidate compounds for activity against target ion channels
US7615356B2 (en) 2000-07-10 2009-11-10 Vertex Pharmaceuticals (San Diego) Llc Ion channel assay methods
AU2003211379A1 (en) * 2002-08-28 2004-03-19 Kinki University Process for producing collagen treated with cysteine protease and collagen treated with cysteine protease
FR2866958B1 (fr) 2004-02-26 2006-08-04 Commissariat Energie Atomique Procede et dispositif de controle du positionnement d'un element biologique sur un support
JP6144255B2 (ja) * 2011-05-13 2017-06-07 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 細胞の選択的トランスフェクション用の光熱基板
AU2014236747B2 (en) 2013-03-15 2019-11-07 The Regents Of The University Of California High-throughput cargo delivery into live cells using photothermal platforms
AU2015235932B2 (en) 2014-03-28 2021-08-05 The Regents Of The University Of California Efficient delivery of large cargos into cells on a porous substrate

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4819647A (en) * 1984-05-03 1989-04-11 The Regents Of The University Of California Intracochlear electrode array
JPH06308118A (ja) * 1993-04-26 1994-11-04 Fujitsu Ltd 細胞の選別と電極刺入方法および装置
IL108775A (en) * 1994-02-25 2003-09-17 Univ Ramot Method for efficient incorporation of molecules into cells
JP3204875B2 (ja) * 1994-06-13 2001-09-04 松下電器産業株式会社 細胞電位測定装置
US5563067A (en) * 1994-06-13 1996-10-08 Matsushita Electric Industrial Co., Ltd. Cell potential measurement apparatus having a plurality of microelectrodes
JPH08122326A (ja) * 1994-10-24 1996-05-17 Fujitsu Ltd 膜電位固定方法及びその装置
JPH09289886A (ja) * 1996-04-25 1997-11-11 Shimadzu Corp 細胞膜電位検出装置

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
Altin, J. G., et al., "Testing the In Vivo Role of Protein Kinase C and c-Fos in Neurite Outgrowth by Microinjection of Antibodies into PC12 Cells," Molecular Biology of the Cell, 3:323-333 (1992).
Bard, A. J., et al., "Chemical Imaging of Surfaces with the Scanning Electrochemical Microscope," Science, 254:68-74 (1991).
Boch, R., et al., "Study of Photoinduced Energy and Electron Transfer in alpha-Terthienyl-Bovine Serum Albumin Conjugates: A Laser Flash Photolysis Study," Photochemistry and Photobiology, 64:92-99 (1996).
Boch, R., et al., "Study of Photoinduced Energy and Electron Transfer in α-Terthienyl-Bovine Serum Albumin Conjugates: A Laser Flash Photolysis Study," Photochemistry and Photobiology, 64:92-99 (1996).
Hamill, O. P., et al., "Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches," Pflügers Archiv, 391:85-100 (1981).
Haydon, P. G., et al., "Membrane Deformation of Living Glial Cells Using Atomic Force Microscopy," Journal of Microscopy, 182:114-120 (1996).
Henriksen G. H., et al., "Laser-Assisted Patch Clamping: A Methodology," Pflügers Archiv, 433:832-841 (1997).
Hoogerwerf, A. C., et al., "A Three-Dimensional Microelectrode Array for Chronic Neural Recording," IEEE Transactions on Biomedical Engineering, 41:1136-1146 (1994).
Horn, R., et al., "Muscarinic Activation of Ionic Currents Measured by a New Whole-Cell Recording Method," J. Gen. Physiol., 92:145-159 (1988).
Jacobs, H. O., et al., "Surface Potential Mapping: A Qualitative Material Contrast in SPM," Ultramicroscopy, 69:39-49 (1997).
Jimbo, Y., et al., "Electrical Stimulation and Recording from Cultured Neurons Using a Planar Electrode Array," Bioelectrochemistry and Bioenergetics, 29:193-204 (1992).
Kovacs, G. T. A., et al., "Regeneration Microelectrode Array for Peripheral Nerve Recording and Stimulation," IEEE Transaction on Biomedical Engineering, 39:893-902 (1992).
Kurata S., et al., "The Laser Method for Efficient Introduction of Foreign DNA into Cultured Cells," Experimental Cell Research, 162:372-378 (1986).
Levitan, E. S., et al., "Neuropeptide Modulation of Single Calcium and Potassium Channels Detected with a New Patch Clamp Configuration," Nature, 348:545-547 (1990).
Marles, R. J., et al., "Thiophenes as Mosquito Larvicides: Structure-Toxicity Relationship Analysis," Pesticide Biochemistry And Physiology, 41:89-100 (1991).
Robinson, D. A., "The Electrical Properties of Metal Microelectrodes," Proceedings of the IEEE, 56:1065-1071 (1968).
Saito et al. "Light does and time dependency of photodynamic cell membrane damage.' Photochemistry and Photobiology. vol. 68 (1998) No. 5, pp. 745-748.* *
Saito et al. ‘Light does and time dependency of photodynamic cell membrane damage.’ Photochemistry and Photobiology. vol. 68 (1998) No. 5, pp. 745-748.*
Thorpe, W. P., et al., "Dynamics of Photoinduced Cell Plasma Membrane Injury," Biophysical Journal, 68:2198-2206 (1995).
Valenzeno, D. P., "Photomodification of Biological Membranes with Emphasis on Singlet Oxygen Mechanisms," Photochemistry and Photobiology, 46:147-160 (1987).

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US20070172600A1 (en) * 2003-04-25 2007-07-26 National Institute Of Information And Communication Technology Incorporated Molecular bonding method and molecular bonding device
US20080014573A1 (en) * 2004-05-26 2008-01-17 Yunisoku Corporation Biosample Manipulation Apparatus
US20090148880A1 (en) * 2004-11-05 2009-06-11 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Method and devices for treating individual biological cells
DE102004053596A1 (de) * 2004-11-05 2006-05-11 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtungen zur Bearbeitung einzelner biologischer Zellen
DE102004053596B4 (de) * 2004-11-05 2006-10-12 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtungen zur Bearbeitung einzelner biologischer Zellen
US8283132B2 (en) 2004-11-05 2012-10-09 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forshung E.V. Method and devices for treating individual biological cells
US20080302674A1 (en) * 2004-12-21 2008-12-11 Kyoto University Probe Unit, Apparatus for Identifying Nucleotide Region and Method of Identifying Nucleotide Region
US20070005117A1 (en) * 2005-06-30 2007-01-04 Fritsch Michael H Extra-cochlear implanted hearing aid device
US8805547B2 (en) 2005-06-30 2014-08-12 Domestic Legacy Limited Partnership Extra-cochlear implanted hearing aid device
US20070225776A1 (en) * 2006-03-22 2007-09-27 Fritsch Michael H Intracochlear Nanotechnology and Perfusion Hearing Aid Device
US7650194B2 (en) * 2006-03-22 2010-01-19 Fritsch Michael H Intracochlear nanotechnology and perfusion hearing aid device
US10119151B2 (en) 2007-07-09 2018-11-06 Brigham Young University Methods and devices for charged molecule manipulation
US8909348B2 (en) 2010-03-30 2014-12-09 Domestic Legacy Limited Partnership Cochlear implant stabilization and positioning device
US20140199765A1 (en) * 2011-05-24 2014-07-17 Brigham Young University Lance device and associated methods for delivering a biological material into a cell

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